Hypertonicity (e.g. high NaCl) activates the esential osmoprotective transcription factor NFAT5 by increasing its abundance, nuclear localization and transactivating activity. It is activated by a network of signaling molecules, whose members we have continued to identify and characterize, as follows: CDK5. In HEK293 cells, mass spectrometry shows phosphorylation of NFAT5-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates NFAT5-T135. Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of NFAT5 but does not affect its transactivating activity. High NaCl induces nuclear localization of NFAT5 faster (<2 h) than it increases its overall protein abundance (>6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear NFAT5 after 16 h. Thus high NaClinduced increase of the overall abundance of NFAT5, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes. c-Abl. High NaCl-induced phosphorylation of NFAT5 at tyrosine-143 was known to be an important factor in increasing its activity in cell culture. We now find that NFAT5 also is phosphorylated at tyrosine-143 in rat renal inner medulla, dependent on the interstitial osmolality. c-Abl seemed likely to be the kinase that phosphorylates NFAT5 because Y143 is in a consensus c-Abl phosphorylation site. We now confirm that, as follows. High NaCl increases c-Abl activity. Specific inhibition of c-Abl by imatinib, siRNA, or c-Abl kinase dead drastically reduces high NaCl-induced NFAT5 activity by reducing its nuclear location and transactivating activity. c-Abl associates with NFAT5 (coimmunoprecipitation)and phosphorylates NFAT5-Y143 both in cell and in vitro. High NaCl-induced activation of ataxia telangiectasia mutated, previously known to contribute to activation of NFAT5, depends on c-Abl activity. Thus, c-Abl is the kinase responsible for high NaCl-induced phosphorylation of NFAT5-Y143, which contributes to its increased activity. PLC-gamma1. Phospholipase C-gamma1 (PLC-gamma1)has a predicted binding site at NFAT5-Y143. We found that: (i) Activation of NFAT5 transcriptional activity by high NaCl is reduced in PLC-gamma1 null cells and in HEK293 cells in which PLC-gamma1 is knocked down by a specific siRNA. (ii) High NaCl increases phosphorylation of NFAT5 at Y143. (iii) Wild-type PLC-gamma1 coimmunoprecipitates with wild-type NFAT5 but not NFAT5-Y143A, and the coimmunoprecipitation is increased by high NaCl. (iv) PLC-gamma1 is part of the protein complex that associates with NFAT5 at its DNA binding site. (v) Knockdown of PLC-gamma1 or overexpression of a PLC-gamma1-SH3 deletion mutant reduces high NaCl-dependent NFAT5 transactivating activity. (vi) Nuclear localization of PLC-gamma1 is increased by high NaCl. (vii) High NaCl-induced nuclear localization of NFAT5 is reduced if cells lack PLC-gamma1, if PLC-gamma1 mutated in its SH2C domain is overexpressed, or if Y143 in NFAT5 is mutated to alanine. (viii) Expression of recombinant PLC-gamma1 restores nuclear localization of wild-type NFAT5 in PLC-gamma1 null cells but not of NFAT5-Y143A. (ix)The PLC-gamma1 phospholipase inhibitor U72133 inhibits nuclear localization NFAT5 but not the increase of its transactivating activity. Thus, when NaCl is elevated, NFAT5 becomes phosphorylated at Y143, resulting in binding of PLC-gamma1 to that site, which contributes to NFAT5 transcriptional activity, transactivating activity, and nuclear localization. MDC1. We used mass spectrometry to analyze proteins that coimmunoprecipitate with NFAT5 in order to identify ones that might contribute to its high NaCl-induced activation. We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in NFAT5 transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with NFAT5 and contribute to its activation by hypertonicity. Thus, MDC1 associates with NFAT5 and contributes to high NaCl-induced increase of that factors transcriptional activity. Phosphatases;SHP-1. Phosphorylation of NFAT5 contributes to its activation. Several of the kinases that are involved were previously identified, but the phosphatases were not. We screened a genomewide human phosphatase siRNA library in human embryonic kidney (HEK)293 cells for effects on NFAT5 transcriptional activity. We found that siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity during normotonicity (290 mosmol/kg) or hypertonicity (500 mosmol/kg, NaCl added)or both.Most siRNAs increase NFAT5 activity, implying that the targeted phosphatases normally reduce that activity. We further studied in detail SHP-1, whose knockdown by its specific siRNA increases NFAT5 transcriptional activity at 500 mosmol/kg. We confirmed that SHP-1 is inhibitory by overexpressing it, which reduces NFAT5 transcriptional activity at 500 mosmol/kg. SHP-1 dephosphorylates NFAT5 at a known regulatory site, Y143, both in vivo and in vitro. It inhibits NFAT5 by both reducing NFAT5 nuclear localization, which is Y143 dependent, and by lowering high NaCl-induced NFAT5 transactivating activity. SHP-1 coimmunoprecipitates with NFAT5 and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP-1 on NFAT5 by increasing phosphorylation of SHP-1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, NFAT5 is extensively regulated by phosphatases, including SHP-1, whose inhibition by high NaCl increases phosphorylation of NFAT5 at Y143, contributing to the nuclear localization and activation of NFAT5. OSM/RAC1. siRNA knockdown of Rac1 reduces high NaCl-induced increase of NFAT5 transcriptional activity by reducing its transactivating activity but not its nuclear localization. Similarly, siRNA knockdown of osmosensing scaffold for MEKK3 (OSM) also reduces high NaCl-dependent NFAT5 transcriptional and transactivating activities. Simultaneous siRNA knockdown of Rac1 and OSM is not additive, indicating a common pathway. The effect of Rac1 on NFAT5 depends on PLC-gamma1. When transfected into PLC-gamma1null mouse embryonic fibroblast cells, catalytically active Rac1 does not increase NFAT5 transcriptional activity unless PLC-gamma1 is reconstituted. Similarly, dominant-negative Rac1 also does not inhibit NFAT5 in PLC-gamma1null cells unless PLC-gamma1 is reconstituted. Thus Rac1/OSM supports NFAT5 activity, mediated via PLC-gamma1.